Bioluminescent markers of neural activity

ABSTRACT

Provided is a polypeptide including an amino acid sequence represented by any of SEQ ID NOs:13-24, or a sequence having at least 90% sequence homology with any one of the foregoing, or a sequence having at least 95% sequence homology with any one of the foregoing. Also provided is a polynucleotide including a fluorescent protein connected to an aequorin by a linker, wherein the amino acid sequence of the fluorescent protein is represented by amino acids 1 through 239 of SEQ ID NO:13, amino acids 1 through 239 of SEQ ID NO:14, amino acids 1 through 237 of SEQ ID NO:15, or amino acids 1 through 237 of SEQ ID NO:16, the amino acid sequence of the linker is represented by amino acids 240 through 256 of SEQ ID NO:13, and the amino acid sequence of the aequorin is represented by amino acids 257 through 448 of SEQ ID NO:13, amino acids 257 through 450 of SEQ ID NO:17, or amino acids 257 through 450 of SEQ ID NO:21. Also provided is a polynucleotide including a sequence that encodes for the polypeptide and a viral vector including the polynucleotide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent application No. 62/684,737, filed Jun. 13, 2019, and U.S. provisional patent application No. 62/684,740, filed Jun. 14, 2019, the entire contents of which are hereby incorporated by reference in their entireties.

GOVERNMENT RIGHTS STATEMENT

This invention was made with Government support under grant number1706761 and 1453339 awarded by the National Science Foundation and grant number EY028391 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing, created on Jun. 1, 2019; the file, in ASCII format, is designated H1482106.txt and is 85.3 KB in size. The file is hereby incorporated by reference in its entirety into the instant application.

FIELD OF THE INVENTION

The subject matter disclosed herein relates to bioluminescent compounds. More particularly, this disclosure relates to calcium-sensitive bioluminescent indicators for reporting neural activity.

BACKGROUND OF THE INVENTION

Certain compounds are capable of emitting luminescence in response to changes of various intracellular ion concentrations. For example, some proteins, in combination with particular compounds within a cell, can emit electromagnetic radiation when complexed with intracellular calcium ions. Because depolarization of a neuron concordant with generation of an action potential entails an elevation in intracellular calcium, such compounds can function as indicators of neural activity, emitting fluorescence from neurons firing an action potential. Potential use of such compounds could include, for example, visualization of activity within neurons of a brain or nerve in vivo or visualizing neural activity of slice preparations or other ex vivo applications.

Brain-machine interfaces (BMIs) allow direct communication to occur between the brain and an external device. BMIs have the potential to restore sensory or motor function to patients with spinal cord injuries or amputations. Preferably, advantageous features of a BMI system would include (1) minimal invasiveness, (2) long lifetime, (3) provision of high information content, (4) robustness, and (5) portability. Conventional BMI systems are deficient in these features. A particular advantageous feature of a BMI system would be if it could differentiate activity of particular neurons within an observed field of neurons, such as when neurons responsive to one input but not another are anatomically co-mingled with neurons of the converse responsiveness, or that do not respond to either of said stimulus, respond to both, or otherwise respond differently to one or more stimuli than do other neurons located in proximity thereto. Conventional BMI systems do not provide such ability to distinguish between activity of neighboring neurons.

A species of jellyfish, Aequorea victoria, produces a protein known as aequorin which is a Ca2+ dependent bioluminescent protein. In jellyfish, aequorin exists as a complex with fluorescent protein (e.g., GFP). In the presence of calcium, aequorin is able to act as a catalytic enzyme in a luciferase reaction. Aequorin oxidizes coelenterazine (CTZ), a molecular cofactor found naturally in jellyfish. The energy gained from the oxidation reaction is passed to the neighboring fluorescent protein which (in the case of GFP) releases a green photon in a process known as chemiluminescence resonance energy transfer (CRET). As a result, neural activity can be reported by aequorin as demonstrated in zebrafish and nerves in the legs of mice. Aequorin-fluorescent protein constructs act as calcium indicators because they have good temporal resolution and are bright with low or no background noise. However, although a single color bioluminescent indicator in the brain can report bulk activity, it cannot distinguish the activity between multiple, intermingled neurons because of the temporal overlap between signals generated from different neurons.

The present disclosure is directed to overcoming these and other deficiencies in the art. As disclosed further herein, use of bioluminescent proteins can provide significant improvement over conventional BMI systems.

SUMMARY OF THE INVENTION

In one aspect, provided is a polypeptide including an amino acid sequence represented by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO: 28, or a sequence having at least 90% sequence homology with any one of the foregoing, or a sequence having at least 95% sequence homology with any one of the foregoing. In another aspect, provided is a polynucleotide including a nucleotide sequence that encodes for the polypeptide. In an embodiment, a viral vector including the polynucleotide is provided.

In another embodiment, provided is a neuron transfected with the polynucleotide. In an example, provided is a neuron transfected with a first of the polynucleotides and a second of the polynucleotides, wherein the first polynucleotide comprises a sequence that differs from the second polynucleotide. In another example, provided is a neuron transfected with a first of the polynucleotides, a second of the polynucleotides, and a third of the polynucleotides, wherein the first polynucleotide comprises a sequence that differs from the second polynucleotide, and the second polynucleotide comprises a sequence that differs from the third polynucleotide.

In another aspect, provided is a plurality of neurons wherein each of the plurality of neurons expresses one or more of the polypeptides having differing amino acid sequences from each other, and a level of expression of a first of the one or more polypeptides relative to a level of expression of a second of the one or more polypeptides in a first neuron differs from a level of expression of the first of the two or more polypeptides relative to a level of expression of the second of the two or more polypeptides in a second neuron.

In another aspect, provided is a plasmid including one or more of the polynucleotides.

In another aspect, provided is a kit including one or more of the polypeptides, one or more polynucleotides, one or more viral vectors, one or more plasmids, or any combination of two or more of the foregoing.

In another aspect, provided is a polypeptide including a fluorescent protein connected to an aequorin by a linker, wherein the amino acid sequence of the fluorescent protein is represented by amino acids 1 through 239 of SEQ ID NO:13, amino acids 1 through 239 of SEQ ID NO:14, amino acids 1 through 237 of SEQ ID NO:15, amino acids 1 through 237 of SEQ ID NO:16, amino acids 1-239 of SEQ ID NO:26, or amino acids 1-476 of SEQ ID NO:28, the amino acid sequence of the linker is represented by amino acids 240 through 256 of SEQ ID NO:13, amino acids 240-300 of SEQ ID NO:26, or amino acids 477-493 of SEQ ID NO:28, and the amino acid sequence of the aequorin is represented by amino acids 257 through 448 of SEQ ID NO:13, amino acids 257 through 450 of SEQ ID NO:17, amino acids 257 through 450 of SEQ ID NO:21, amino acids 381-488 of SEQ ID NO:26, or amino acids 494-685 of SEQ ID NO 28. Also provided is a polynucleotide including a sequence that encodes for the polypeptide. In an example, provided is a viral vector including the polynucleotide.

In another embodiment, provided is a neuron transfected with the polynucleotide. In an example, provided is a neuron transfected with a first of the polynucleotides and a second polynucleotides, wherein the first polynucleotide comprises a sequence that differs from the second polynucleotide. In another example, provided is a neuron transfected with a first of the polynucleotides, a second of the polynucleotides, and a third of the polynucleotides, wherein the first polynucleotide comprises a sequence that differs from the second polynucleotide, and the second polynucleotide comprises a sequence that differs from the third polynucleotide.

In another aspect, provided is a plurality of neurons wherein each of the plurality of neurons expresses one or more of the polypeptides having differing amino acid sequences from each other, and a level of expression of a first of the one or more polypeptides relative to a level of expression of a second of the one or more polypeptides in a first neuron differs from a level of expression of the first of the one or more polypeptides relative to a level of expression of the second of the one or more polypeptides in a second neuron.

In an example, the polynucleotide is a plasmid.

Also provided is a kit including one or more of the polypeptides, one or more of the polynucleotides, one or more of the viral vectors, one or more of the plasmids, or any combination of two or more of the foregoing.

Also provided is a method of detecting neural activity in different neurons including inducing expression of two or more of the polypeptides having differing amino acid sequences from each other in two or more neurons, wherein a level of expression of a first of the two or more polypeptides relative to a level of expression of a second of the two or more polypeptides in a first neuron differs from a level of expression of the first of the two or more polypeptides relative to a level of expression of the second of the two or more polypeptides in a second neuron, applying coelenterazine (CTZ) or 2-deoxycoelenterazine (CTZ-H) to the two or more neurons, inducing activity in one or more of the two or more neurons, and detecting bioluminescence emitted by one or more of the neurons.

In an embodiment, one of the neurons emits bioluminescence of a first wavelength when stimulated and another of the neurons emits bioluminescence of a second wavelength when stimulated. In another embodiment, inducing expression comprises transfecting each of the two or more neurons with one or more of two or more of the polynucleotides wherein two or more of the two or more polypeptides have differing sequences from each other. In another embodiment, transfecting includes contacting the two or more neurons with two or more of the viral vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:

FIG. 1 shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for SEQ ID NO:13.

FIG. 2A and FIG. 2B show a rise/decay plot in response to saturating calcium for SEQ ID NO:13.

FIG. 3 shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with a sequence corresponding to SEQ ID NO:14.

FIG. 4 shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with an amino acid sequence corresponding to SEQ ID NO:15.

FIG. 5 shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with an amino acid sequence corresponding to SEQ ID NO:16.

FIG. 6A shows a composite of bioluminescent spectra for polypeptides with amino acid sequences corresponding to SEQ ID NOs:13-16 and 26.

FIG. 6B shows relative light output normalized to protein concentration for SEQ ID NOs:13-16 and 26, expressed as a percentage output of SEQ ID NO:26.

FIG. 7A shows a two-photon excited fluorescence (2-PEF) microscopy image of neurons expressing different bioluminescent protein constructs as disclosed herein and FIG. 7B is a 3-dimensional plot showing RGB intensity of neurons shown in FIG. 7A.

FIG. 8A shows histological data of mouse barrel cortex following transduction of neurons expressing multiple different bioluminescent proteins and FIG. 8B shows RGB intensity levels of 15 neurons from FIG. 8A.

FIG. 9 shows a 2-PEF microscopy image of neurons in the mouse cortex expressing different color bioluminescent proteins.

FIG. 10 shows stimulus-triggered neural activity in mouse barrel cortex neurons following transfection with AAV vectors driving expression of three different bioluminescent proteins.

FIG. 11 shows light emission of three different colors in 1-second epochs before, during, and after shock to the whisker pad with electrodes in two different locations (a and b) in mouse barrel cortex following injection with AAV vectors driving expression of three different bioluminescent proteins.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure relates to compositions, methods, systems, and kits for inducing cells such as neurons to emit detectable electromagnetic radiation, including having wavelengths of visible light, when activated and experiencing elevated calcium levels intracellularly. Compositions disclosed herein could likewise be used for identifying elevations of calcium levels intracellularly in other cells such as myocytes such as cardiomyocytes, or other cell types. Such compositions could also be used for measuring changes in extracellular calcium levels, or in solutions without whole cells.

Bioluminescence imaging (BLI) is a form of optical imaging that utilizes the detectable electromagnetic radiation, such as visible light, produced during luciferase-mediated oxidation of substrates to track processes at a molecular level. Molecular imaging with bioluminescence is advantageous because it is both non-invasive and has high signal-to-noise ratios because mammalian tissue has low intrinsic bioluminescence. As disclosed herein, neurons may be manipulated to express bioluminescent proteins that bioluminesce in response to elevated intracellular calcium levels. Furthermore, different species of bioluminescent molecules disclosed herein emit differing wavelengths of electromagnetic radiation. Thus, a neuron expressing one bioluminescent molecule would emit one wavelength of electromagnetic radiation in response to elevated calcium levels, while another neuron expressing a different bioluminescent molecule, or the same bioluminescent molecule in combination with one or more different bioluminescent molecule, would emit electromagnetic radiation of a different frequency upon elevation of intracellular calcium.

Surprisingly, as further disclosed herein, inducing expression of different combinations and/or different levels of bioluminescent, calcium-responsive molecules in different neurons gives rise to an exponential increase in the different emission spectra exhibited from cell to cell in response to calcium influx. That is, when inducing the expression of different levels of a bioluminescent molecule and/or different bioluminescent molecules in different combinations from one cell to the next, and or with differing levels of relative expression between bioluminescent molecules expressed per cell, each cell emits at a net frequency reflective of the particular bioluminescent molecule(s) it expresses and at a given level of expression. With, for example, six different bioluminescent markers applied to a field of neurons, at least 3,000 or more distinct emission spectra can be observed from different cells in response to calcium elevations owing to stochastically-determined differences in which bioluminescent molecules become expressed and at what relative levels to one another per cell. The wide variety of differentiable spectra obtained was particularly surprising in view of the failure of other, conventional technologies (e.g., BRAINBOW technology) to generate such diversity when cells are induced to express different marker compounds and/or at different levels. The success of the combinatorial approach undertaken as disclosed herein in generating a wide diversity of emission spectra per cell represents a significant advance over conventional technology that would not have been predicted on the basis of previous attempts with different markers.

Thus, as disclosed herein, multiple bioluminescent molecules may be applied simultaneously across a field of neurons such that neurons express different bioluminescent molecules at relative levels that differs stochastically from neuron to neuron. For example, two, three, four, five, six, or more bioluminescent markers with emission spectra that differ from each other may be applied across a field of neurons, and different neurons express different levels of each of the one, two, three, four, five, six, or more bioluminescent markers compared to other neurons in the field or with which each neuron is spatially comingled. As a result, each neuron would emit in the presence of elevated intracellular calcium at an emission frequency that is a reflection of the combined emission spectrum of each of the one, two, three, four, five, six, or more bioluminescent markers it is expressing at the level at which it is expressing each thereof. With different neurons stochastically expressing different relative levels of the two, three, four, five, or more bioluminescent marks compared to the other neurons within the field, each neuron would fluoresce at a different frequency than the other neurons upon elevations of intracellular calcium. Thus, activity of one neuron as opposed to another could be distinguished not just by the location of the neuron emitting bioluminescence but also by the frequency of bioluminescence it is emitting.

As disclosed herein, where cells such as neurons are described as exhibiting different levels of expression of two or more bioluminescent markers such a bioluminescent polypeptides, an example is where a neuron may express none of either of two markers, or some of only one marker, or some of only another marker, or some of any two or more of the two or more markers (such as of up to six or more). Thus, in some examples, a field of neurons may be induced to express from one to six or more bioluminescent proteins. Some neurons of the field may express only one of the six. For example, six different neurons may each express only one of the six markers, and each a different marker than the other of those six. Other neurons may express two or more makers, in any combination. This non-limiting example may include a plurality of neurons wherein each of the plurality of neurons expresses one or more bioluminescent markers having differing amino acid sequences from each other, and a level of expression of a first of the one or more polypeptides relative to a level of expression of a second of the one or more polypeptides in a first neuron (for example, a neuron that expresses some of one marker and none of any other) differs from a level of expression of the first of the one or more polypeptides relative to a level of expression of the second of the one or more polypeptides in a second neuron (which may express some of each of two or more markers, or some of one marker not expressed by the first neuron and some of a marker not expressed in the first neuron). Other neurons of the field may each express more than none of any combination of at least two of the bioluminescent markers, which may also include a first neuron and a second that express different relative levels of two or more bioluminescent markers than each other, or than any neuron that expresses some of only one but not of any other bioluminescent marker.

Expression of bioluminescent compounds may be accomplished by any known method. For example, cells can be transfected with nucleotide sequences (DNA, RNA, etc.) that encode for bioluminescent proteins as disclosed herein. As used herein, transfecting, transfected, or transfection includes any process for introducing nucleic acids or proteins into cells, including transduction through viral vector-mediated gene transfer. For example, adeno-associated viral (AAV) vectors may be used to transfect neurons, with different vectors driving expression of genetic material encoding different bioluminescent proteins. AAV vectors derived from a particular serotype or from mixed serotypes may be adopted and used depending on the particular application. If a population of neurons is contacted with a solution or solutions containing different viral vectors such as these, each containing material to drive the expression of different bioluminescent proteins, different cells can be transfected with stochastically different levels of payload from different vectors and thus express relative levels of bioluminescent proteins that differ inter-neuronally. In other examples, different viral vectors may be used, such as lentiviruses, retroviruses, HSV-viral vectors, or other viral vectors known to be effective at driving protein expression in neurons. In some examples, combinations of viral vectors may be used, rather than only one type of vector used to drive expression of all bioluminescent proteins.

Other transfection methods may also be used to drive expression, such as lipofection, electroporation, microinjection, gene gun, continuous infusion, and sonication, impalefection, hydrostatic pressure transfection, with genetic material that drives expression of the bioluminescent protein. In other examples, bioluminescent proteins themselves may be introduced into cells rather than genetic material driving their production by cells. All such examples are considered methods of promoting expression of a bioluminescent composition or compositions in cells, and may be used in combination with one another. In other examples, an extracellular bioluminescent compound or compounds may be used such as by addition of a bioluminescent polypeptide or polypeptides directly to a solution, for indicating elevated calcium levels therein.

A bioluminescent composition in accordance with the present disclosure may be a polypeptide including a fluorescent protein connected to an aequorin by a linker. For example, the fluorescent protein may be N-terminal to the linker and the aequorin may be C-terminal to the linker. In other examples, the aequorin may be N-terminal and the fluorescent protein may be C-terminal. In some examples, there may not be additional amino acids between a fluorescent protein and a linker, or between a linker and an aequorin, or neither between a fluorescent protein and a linker nor between a linker and an aequorin. Polynucleotides encoding for such polypeptides are also disclosed herein. As would be appreciated, because of the degeneracy of the genetic code, many different polynucleotide sequences could drive expression of a given bioluminescent polypeptide disclosed herein, and all such sequences for all such bioluminescent polypeptides are explicitly included in the present disclosure. Such a polynucleotide may exist in a form convenient for storage or expression within a cell such as a plasmid. Such a plasmid may contain an origin of replication, transcriptional start sites, and/or other structural features that promote replication thereof in a host cell and/or production of and/or translation from a transcript to promote expression of a bioluminescent polypeptide.

Examples of fluorescent proteins include mCerulean (represented by amino acids 1-239 of SEQ ID NO:13), eCFP (represented by amino acids 1-239 of SEQ ID NO:14), mTagBFP2 (represented by amino acids 1-237 of SEQ ID NO:15), mTFP1 (represented by amino acids 1-237 of SEQ ID NO:16), eGFP (represented by amino acids 1-239 of SEQ ID NO:26), and tdTomato (represented by amino acids 1-476 of SEQ ID NO:28). Examples of linkers are represented by amino acids 240-256 of SEQ ID NO:13, amino acids 240-300 of SEQ ID NO:26, and amino acids 477-493 of SEQ ID NO:28. Other examples may include a linker as a portion of an amino acid sequence linking a fluorescent protein to an aequorin wherein the linker has an amino acid sequence that differs from the foregoing examples. In some nonlimiting examples, the linker may be from between 10 and 100 amino acids in length. Examples of aequorins include aequorin from Aequorea victoria (represented by amino acids 257-448 of SEQ ID NO:13), obelin from Obelia longissima (represented by amino acids 257-450 of SEQ ID NO:17), and aequorin from Aequorea macrodactyla (represented by amino acids 257-450 of SEQ ID NO:21), referred to herein as AMac. All combinations of any one of the foregoing fluorescent proteins with any one of the foregoing aequorins connected by a linker are included in the present disclosure. A non-limiting list of examples of such combinations includes polypeptides with amino acid sequences represented by SEQ ID NOs:13-24, 26, and 28. All polynucleotides that encode for any bioluminescent protein disclosed herein is also explicitly included in the present disclosure. A non-limiting list of examples includes polynucleotides with sequences represented by SEQ ID NOs:1-12 (which encode for the bioluminescent proteins with amino acid sequences corresponding to SEQ ID NOs:13-24, respectively), and SEQ ID NOs:25 and 27 (which encode for the bioluminescent proteins with amino acid sequences corresponding to SEQ ID NOs:26 and 28, respectively.

In some examples, a bioluminescent polypeptide may include a phosphoprotein that differs by one or more amino acids from mCerulean (as represented by amino acids 1-239 of SEQ ID NO:13), eCFP (as represented by amino acids 1-239 of SEQ ID NO:14), mTagBFP2 (as represented by amino acids 1-237 of SEQ ID NO:15), mTFP1 (represented by amino acids 1-237 of SEQ ID NO:16) , eGFP (represented by amino acids 1-239 of SEQ ID NO:26), or tdTomato (represented by amino acids 1-476 of SEQ ID NO:28). For example, a phosphoprotein segment of a bioluminescent protein may differ by up to 10% in amino acid sequence, or by up to 5% in amino acid sequence, or by between 10% and 5% or between 5% and 0% from the foregoing specific examples. A linker segment of a bioluminescent phosphoprotein may also differ by 10%, 5%, between 10% and 5%, or between 5% and 0% from amino acids 240-256 of SEQ ID NO:13, amino acids 240-300 of SEQ ID NO:26, and amino acids 477-493 of SEQ ID NO:28. Furthermore, an aequorin segment of a bioluminescent phosphoprotein may differ in amino acid content by 10%, 5%, between 10% and 5%, or between 5% and 0% from amino acids 257-448 of SEQ ID NO:13, amino acids 257-450 of SEQ ID NO:17, or amino acids 257-450 of SEQ ID NO:21. A bioluminescent protein may differ by 10%, 5%, between 10% and 5%, or between 5% and 0%, from any one of SEQ ID NOs:13-24, 26, or 28. All polynucleotide sequences that may encode for any of the foregoing segments of bioluminescent proteins or bioluminescent proteins are explicitly disclosed herein. For example, a nucleotide sequence may differ by 10%, 5%, between 0% and 5%, or between 5% and 0% in nucleotide sequence from SEQ ID NOs.1-12, 25, or 27.

Any fluorescent protein may be linked to any aequorin by any linker disclosed herein. Some neurons may be induced to express a bioluminescent marker of activity with one fluorescent protein linked to a given aequorin by a linker, whereas another neuron may be induced to express a bioluminescent marker of activity with the same fluorescent protein and linker but a different aequorin, or the same linker and aequorin but a different fluorescent protein, or the same fluorescent protein and aequorin but a different linker. In other cases two neurons may be induced to express bioluminescent markers of activity with the same fluorescent protein as each other but a different linker and different aequorin, or the same linker as each other but a different fluorescent protein and a different aequorin, or the same aequorin as each other but a different fluorescent protein and a different linker. All possible combinations of al fluorescent proteins, linkers, and aequorins disclosed herein are explicitly contemplated and included in the present disclosure. In this manner, neurons may be independently induced to express bioluminescent markers of activity that have no, only one, only two, or three of a fluorescent protein, linker, and/or aequorin in common with each other.

Neurons may be contacted with a coelenterazine molecule together with a bioluminescent protein or upon induced expression thereof. For example, coelenterazine or 2-deoxycoelenterazine may be applied to neurons transfected with a polynucleotide sequence that drives expression of a bioluminescent protein disclosed herein. In the presence of calcium, such as when intracellular calcium levels are elevated in conjunction with a depolarization event, aequorin may oxidize a coelenterazine molecule or analog thereof (e.g., 2-deoxycoelenterazine), whereupon the energy gained from the oxidation reaction is passed to the fluorescent protein segment of the bioluminescent protein inducing photon release thereby by CRET. Depending on the combination of aequorin and fluorescent protein segments used, and different combinations of expression of various bioluminescent proteins comprising such various segments, different neurons may emit different wavelengths of fluorescence when exposed to elevated calcium levels.

For detecting and measuring fluorescence emitted by neurons expressing a bioluminescent protein as disclosed herein, various known optical imaging devices and techniques may be used. Charge coupled device (CCD) cameras, for example, are imaging modalities that spatially encode incident photons and their intensity into an image. Integrating photon counts on a CCD camera, however, may decrease temporal resolution. To increase temporal resolution to spatially localize neurons, photodetectors (for example, photon counting multiplier tubes (PMTs), photodiodes, or CCD/CMOS detector technologies) may be used. In an example, PMTs convert photonic signals into a current which decreases spatial resolution since it diffusely collects scattered photons. However, by color-coding cells such as neurons with different bioluminescent compositions and/or different combinations of compositions to permit inter-neuronal differentiation of the frequency of electromagnetic radiation emitted in response to elevated intracellular calcium, different color indicators and sampling with spectrally separated PMTs, a unique spectrum of colors that provide spatial resolution is obtained as disclosed herein. With a high sampling rate, PMTs are able to collect photons from hundreds of neurons that may be firing. Recording equipment may be sensitive to a wide range of frequency of electromagnetic radiation, within the visual spectrum, or outside thereof, depending on emission spectra of bioluminescent proteins used for signaling neural activity.

Such recording device or system may be connected to a computer for recording an analyzing bioluminescence emitted from a given population or field of neurons being measured. Recordings may be recorded on standard computer media systems and/or analyzed by software for depicting and analyzing activity of neurons within a recording field in response to different types of stimuli (e.g., sensory stimuli, pharmacological stimuli, electrical stimuli, etc.).

In an embodiment, bioluminescent proteins, polynucleotides encoding therefore, viral vectors or plasmids including such polynucleotides, or any combination thereof, may be included in a kit for administration to neurons for purposes of measuring differential activity as disclosed herein. For example, such a kit may have a combination of one or more, two or more, three or more, four or more, five or more, six or more, or higher numbers of bioluminescent proteins, or plasmids or viral vectors containing polynucleotides encoding therefore, or any combination of the foregoing, in a usable form (e.g., lyophilized, or in a buffered solution, etc.) for administration by a researcher or clinician to neurons, nerves, or a brain of a subject for purposes of transfecting neurons with differential relative levels of expression of the bioluminescent markers included or encoded for by polynucleotides included in the kit. A kit may also contain additional reagents, buffers, solutions, other transfection reagents, or compounds (such as CTZ or CTZ-H) for application in use of the bioluminescent polypeptides, polynucleotides, or viral vectors.

Bioluminescent polypeptides or polynucleotides encoding therefor or viral vectors for driving the expression thereof as disclosed herein may be used in neurons taken from a subject of any intended species for ex vivo use or applied in vivo to a nervous system of a subject of any given species of interest. For example, with appropriate promoter sequences and transcriptional features for a given target species, they could be adopted for use in invertebrates or vertebrates. For example, they could be used in insects, fish, amphibians, reptiles, birds, or mammals. For example, they could be used in rodent, ungulate, canine, feline, leporine, porcine, primate, or other species. For example, they could be used in mice, rats, humans, or non-human primates. As disclosed herein, bioluminescent proteins or vectors for driving their expression may be injected or otherwise applied to a nervous system of such species then bioluminescence of the treated area observed in response to stimulatory input known or believed to recruit activity of neurons in such region, or hypothesized or suspected of doing so.

EXAMPLES

The following examples are intended to illustrate particular embodiments of the present disclosure, but are by no means intended to limit the scope thereof.

FIG. 1 shows bioluminescent spectra of a polypeptide with an amino acid sequence corresponding to SEQ ID NO:13 sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H.

FIG. 2A and FIG. 2B show kinetic rise/decay data collected on SFM-400 stopped flow mixer in response to saturating calcium. A polypeptide with an amino acid sequence corresponding to SEQ ID NO:13 was incubated overnight in CTZ-H, buffer exchanged to zero calcium buffer (to remove excess coelenterazine), and rapidly mixed with calcium to elucidate kinetic response (FIG. 2B is an axis zoom of FIG. 2A over the first 0.5 sec).

FIG. 3 shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with a sequence corresponding to SEQ ID NO:14.

FIG. 4 shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with an amino acid sequence corresponding to SEQ ID NO:15.

FIG. 5 shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with an amino acid sequence corresponding to SEQ ID NO:16.

FIG. 6A shows a composite of bioluminescent spectra for polypeptides with amino acid sequences corresponding to SEQ ID NOs:13-16 and 26, illustrating differentiation of emission spectra amongst these examples. Cells expressing one or combinations of these bioluminescent proteins can be distinguished by their differences in emission spectra in response to calcium elevations. Spectra were sampled in response to saturating Ca2+ (200 mM) at room temperature with 100 ms integration (Ocean Optics QE65000) after overnight incubation with Coelenterazine-H. As shown in FIG. 6A, bioluminescent proteins as disclosed herein (e.g., those represented by SEQ ID NOs:13-16) showed notable blue-shifting relative to other bioluminescent proteins, such as with a sequence indicated by SEQ ID NO:26. Such blue-shifting was surprising, and not fully explained by Förster or other stable methods in Förster resonance energy transfer pairs. Without being limited to a particular function, one suggested explanation for such unexpected bleu-shifting that different variations in amino acid sequence amongst fusion pairs influences biophysical properties to blue-shift emission spectra.

FIG. 6B Shows relative light output normalized to protein concentration for SEQ ID NOs.13-16 and 26. Light output is calculated as mean gray value per frame collected with Basler AG CMOS camera; average of three trials. Total light collected over entire time course is stated as a (%) relative to SEQ ID NO:26. As disclosed herein, bioluminescent proteins having amino acid sequences represented by, for example, SEQ ID NOs:13-16 showed greater light emission relative to that of SEQ ID NO:26, indicated by their relatively higher areas-under-curve as indicated in FIG. 6B.

Viral Vector Injection for Indicator Expression

Individual strains of adeno-associated virus (AAV) viral vectors were packaged with a polynucleotide encoding a single color bioluminescent protein construct. In a sterile surgical preparation, the mouse is fixed onto a stereotax. A drill bit is used to create a burr hole in the skull to expose the surface of the brain. AAV vectors packed with individual polynucleotides encoding a single color bioluminescent protein construct are either individually or mixed with other colors and injected with a glass pipette into the rodent barrel cortex which is 3.5 mm lateral to bregma and 1.5 mm posterior to bregma. A bolus (100 nL to 1000 nL) is injected about 400 μm in depth. The mouse is sutured and following chronic protocols, administered drugs to facilitate healing and prevent inflammation.

In Vivo Imaging and Bioluminescence Measurement

After approximately three weeks during which time neurons are transduced to express bioluminescent proteins, an acute imaging experiment was performed. In an acute surgical preparation, a craniotomy about ˜3 mm in diameter was performed, centered over the barrel cortex. Coelenterazine, the cofactor required for bioluminescence, is injected with a glass pipette into the barrel cortex, in the same region that the virus was previously injected into. Coelenterazine is also applied topically and gel foam is placed over the surface of the brain to prevent the surface from drying out. In a dark room, coelenterazine is allowed approximately one hour to reconstitute. The mouse is then brought into the imaging facility and an electric stimulator is attached to the whisker pad. Using a non-imaging modality, augmented from optics typically used in 2-photon excitation fluorescence microscopy (2-PEF), light emitted from the rodent brain in response to stimulus is spectrally separated and detected on photomultiplier tubes (PMTs).

Tissue Processing and Histology

In order to prepare the tissue for histology, the mouse is perfused via a transcardial perfusion. At the conclusion of an imaging experiment, brain tissue is removed and immersed in fixative. The fixed brain is sliced into 70 μm slices and mounted onto slides for 1-photon imaging.

Results

Mixing and co-injecting multiple AAV vectors, each encoding a single color, produced stochastic and varied transduction profile in cortical neurons

Both individual and mixes of AAV vectors packaged with different bioluminescent proteins that bioluminesce at different frequencies in response to elevated calcium levels were injected into mouse models to test for expression amounts and patterns. For mixtures of colors, each vector contained 2×10{circumflex over ( )}11 GC/mL. Injecting a mixture of colors resulted in individual cells with distinct spectral patterns. Shown in FIG. 7A is a two-photon excited fluorescence (2-PEF) microscopy image of mouse cortex showing fluorescence of transfected cells in response to fluorescent stimulation (as opposed to activity-induced calcium increase), demonstrating that different cells emitted different frequencies of florescence due following transfection due to stochastically differential expression in different neurons of bioluminescent proteins. FIG. 7B shows a 3D plot of RGB intensity of neurons shown in FIG. 6A demonstrating their differential emission levels.

In the example in FIG. 8A, a mouse was co-injected with four different AAV vectors with different bioluminescent proteins and the neurons were allowed 3 weeks to transduce. An acute craniotomy was performed and stimulus triggered bioluminescent data was acquired. The mouse was then perfused and histology was performed to reveal the expression pattern. Neurons were identified as regions with higher intensities. The image shows that neurons are uniquely and stochastically labeled. The ratio of the fluorescence from different color indicators in each cell is displayed in FIG. 8B. The ratios of three different emission channels measured within neurons varied between cells, with several cells showing distinct patterns in color.

Neural Activity-Dependent Bioluminescence in a Seizure Model.

Applying pentylenetetrazole (PTZ) topically induces a seizure in the mouse cortex that can be recorded optically, demonstrating for the first time that the mouse brain can fluoresce from stimulated neural activity. PTZ causes chemically induced seizures. During seizure activity, the bioluminescent protein constructs released photons that were collected by the PMTs in the presence of coelenterazine. For the first time, it was shown that the rodent brain could emit in vivo detectable bioluminescence from neural activity.

Somatosensory Stimulation-Evoked Neural Activity Reported by Multicolor Bioluminescence

In another anesthetized mouse, a stimulation of the whisker pad produced light emissions at multiple wavelengths. Approximately three weeks after the virus injection, an acute craniotomy was performed on the mouse to reveal the surface of the barrel cortex, where three AAV vectors driving expression of different bioluminescent protein had been injected. A 2-PEF microscopy image of neurons in the mouse cortex expressing different color bioluminescent proteins is shown in FIG. 9. Coelenterazine was injected and allowed to reconstitute within the brain at which point an electric stimulator was attached to the mouse whisker pad. Stimulating the whisker pad at 10 Hz resulted in neural activity in the barrel cortex, as indicated by bioluminescent signals collected. Across a number of trials, the same stimulation of the whisker pad resulted in emissions of light with similar temporal and spectral shapes (FIG. 10).

Relocating the location of stimulus on the whisker pad resulted in different patterns of neural activity that was repeatable

For measuring stimulation-evoked activity, stimulus electrodes were initially placed on one area of the whisker pad. The stimulus electrodes were then moved to a slightly different position on the whisker pad and the same shock stimulus was applied again. In both instances, when the intensity of each emission channel is plotted on a separate axis, the emission from one second epochs before, after, and during the stimulus resulted in clearly different color combinations. As expected, moving the area of stimulation on the whisker pad resulted in different spectral and temporal light emissions. Shown in FIG. 11 is light emission in 1-second epochs before, during, and after shock to the whisker pad with electrodes in two different locations (a and b). Each trace is a stimulus triggered average of 5 trials where the intensity in the three different emission channels is plotted as a single point and sequential points are connected. Moving the area of stimulus (a) to another area of stimulus (b) affected the patterns of neural activity in the barrel cortex.

Methods

Surgical Procedure for Burr Hole Injections

A drill is mounted into a micromanipulator over the field. A micropipette loaded with a viral vector for driving expression of bioluminescent polypeptide is loaded into a micromanipulator. Mice are anesthetized under 5% isoflurane at the site of the drill and maintained at 1.5-2% isoflurane in 100% oxygen for the duration of the surgery. Mice are injected with ketoprofen (2 mg/mL at 2.5 uL/g of mouse) and dextamethasone (0.1 mg/mL at 2 uL/g of mouse) subcutaneously. Then mice are injected with atropine sulfate (0.15 mg/mL at 3.3 uL/g of mouse) intramuscularly. Mice are then mounted on a stereotax for intracranial injection. Bupivacaine (0.125%) may be injected subcutaneously over the incision site.

Holes are drilled in the exposed skull at the pre-determined stereotaxic coordinates to expose barrel cortex. Viral vectors are then applied to the exposed cortical tissue via micropipette, at a depth of, for example, ˜500 um deep. Mice are then sutured and recovered from anesthesia. Transduction is allowed to occur for the next 14-21 days before imaging.

Imaging and recording of bioluminescence is performed 21 days after injections to allow for expression of the bioluminescent protein. Skin over the injection site is removed and skull exposed. An approximately 4 mm diameter circle is drilled around the injection site and the skull flap removed. Gel foam is wet with coelenterazine and placed over surface of the brain to reconstitute for at least an hour, prior to imaging. Electric stimulus is then attached to the whisker pad region of the mouse and bioluminescence detected by PMT apparatus in response to various trains of stimulation.

Histology

Mice were perfused transcardially immediately following imaging session with 1×PBS and 4% Paraformaldehyde (PFA). Brains were removed and stored in 4% PFA for an additional 24 hours. Brains were transferred into 30% sucrose for at least 24 hours, or until the brain is saturated with the 30% sucrose, then repeated with 30% sucrose. Slices at 70 um thickness were taken and stored on Superfrost cover glass and imaged underneath 1-photo microscope and images captured with a fluorescent camera.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the present disclosure and these are therefore considered to be within the scope of the present disclosure as defined in the claims that follow. 

1. A polypeptide comprising an amino acid sequence represented by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or a sequence having at least 90% sequence homology with any one of the foregoing.
 2. The polypeptide of claim 1, comprising a sequence having at least 95% sequence homology with SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24.
 3. The polypeptide of claim 1, comprising a sequence having at least 99% sequence homology with SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24.
 4. The polypeptide of claim 1, comprising an amino acid sequence represented by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:0, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24.
 5. A polynucleotide comprising a nucleotide sequence that encodes for the polypeptide of claim
 1. 6. A viral vector comprising the polynucleotide of claim
 5. 7. A neuron transfected with the polynucleotide of claim
 5. 8. A neuron transfected with a first polynucleotide of claim 5 and a second polynucleotide of claim 5, wherein the first polynucleotide comprises a sequence that differs from the second polynucleotide.
 9. A neuron transfected with a first polynucleotide of claim 5, a second polynucleotide of claim 5, and a third polynucleotide of claim 5, wherein the first polynucleotide comprises a sequence that differs from the second polynucleotide, and the second polynucleotide comprises a sequence that differs from the third polynucleotide.
 10. A plurality of neurons wherein each of the plurality of neurons expresses one or more polypeptides of claim 1 having differing amino acid sequences from each other, and a level of expression of a first of the one or more polypeptides relative to a level of expression of a second of the one or more polypeptides in a first neuron differs from a level of expression of the first of the one or more polypeptides relative to a level of expression of the second of the one or more polypeptides in a second neuron.
 11. A plasmid comprising a polynucleotide of claim
 5. 12. (canceled)
 13. A polypeptide comprising a fluorescent protein connected to an aequorin by a linker, wherein the amino acid sequence of the fluorescent protein is represented by amino acids 1 through 239 of SEQ ID NO:13, amino acids 1 through 239 of SEQ ID NO:14, amino acids 1 through 237 of SEQ ID NO:15, or amino acids 1 through 237 of SEQ ID NO:16, the amino acid sequence of the linker is represented by amino acids 240 through 256 of SEQ ID NO:13, and the amino acid sequence of the aequorin is represented by amino acids 257 through 448 of SEQ ID NO:13, amino acids 257 through 450 of SEQ ID NO:17, or amino acids 257 through 450 of SEQ ID NO:21.
 14. A polynucleotide comprising a sequence that encodes for the polypeptide of claim
 13. 15. A viral vector comprising the polynucleotide of claim
 14. 16. A neuron transfected with the polynucleotide of claim
 14. 17. A neuron transfected with a first polynucleotides of claim 14 and a second polynucleotide of claim 14, wherein the first polynucleotide comprises a sequence that differs from the second polynucleotide.
 18. A neuron transfected with a first polynucleotides of claim 14, a second polynucleotide of claim 14, and a third polynucleotide of claim 14, wherein the first polynucleotide comprises a sequence that differs from the second polynucleotide, and the second polynucleotide comprises a sequence that differs from the third polynucleotide.
 19. A plurality of neurons wherein each of the plurality of neurons expresses one or more polypeptides of claim 13, and a level of expression of a first of the one or more polypeptides relative to a level of expression of a second of the one or more polypeptides in a first neuron differs from a level of expression of the first of the one or more polypeptides relative to a level of expression of the second of the one or more polypeptides in a second neuron.
 20. A plasmid comprising a polynucleotide of claim
 14. 21. (canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled) 